Germacrene B – a central intermediate in sesquiterpene biosynthesis

Germacranes are important intermediates in the biosynthesis of eudesmane and guaiane sesquiterpenes. After their initial formation from farnesyl diphosphate, these neutral intermediates can become reprotonated for a second cyclisation to reach the bicyclic eudesmane and guaiane skeletons. This review summarises the accumulated knowledge on eudesmane and guaiane sesquiterpene hydrocarbons and alcohols that potentially arise from the achiral sesquiterpene hydrocarbon germacrene B. Not only compounds isolated from natural sources, but also synthetic compounds are dicussed, with the aim to give a rationale for the structural assignment for each compound. A total number of 64 compounds is presented, with 131 cited references.


Introduction
Terpenoids constitute the largest class of natural products with ca. 100,000 known compounds. Biosynthetically, all terpenoids are derived from only a few acyclic precursors, including the monoterpene precursor geranyl diphosphate (GPP) [1], the precursor for sesquiterpenes farnesyl diphosphate (FPP) [2], geranylgeranyl diphosphate (GGPP) towards diterpenes [3], and the sesterterpene precursor geranylfarnesyl diphosphate (GFPP) [4]. It has been demonstrated recently, that even farnesylfarnesyl diphosphate (FFPP) can serve as a precursor to triterpenes [5], a compound class that was believed to be solely derived from squalene. Terpene synthases convert these linear precursors through cationic cascade reactions into terpene hydrocarbons or alcohols [6][7][8]. For type I terpene synthases this multistep process is initiated by the abstraction of diphosphate to produce an allyl cation that subsequently undergoes typical cation reactions such as cyclisations by intramolecular attack of an olefin to the cationic centre, Wagner-Meerwein rearrangements, hydride or proton shifts. The process is terminated by deprotonation to yield a terpene hydrocarbon or by nucleophilic attack of water to generate a terpene alcohol.
For the precursor of sesquiterpenes FPP six initial cyclisation modes are possible (Scheme 1). After ionisation to A either a 1,10-cyclisation to the (E,E)-germacradienyl cation (B) or a 1,11-cyclisation to the (E,E)-humulyl cation (C) is possible. Reattack of diphosphate at C-3 results in nerolidyl diphosphate (NPP) that can undergo a conformational change by rotation around the C-2/C-3 single bond, which allows reionisation to D. This intermediate can react in a 1,10-cyclisation to the (Z,E)-germacradienyl cation (E) or a 1,11-cyclisation to the (Z,E)-humulyl cation (F), the E/Z stereoisomers of B and C. Furthermore, a 1,6-cyclisation to the bisabolyl cation (G) or a 1,7-cyclisation to H may follow, which is not possible from A because of its 2E configuration (a hypothetical (E)-cyclohexene or (E)-cycloheptene would be too strained, the smallest possible ring with an E configuration is (E)-cyclooctene).
In some cases the initially formed neutral product can become reprotonated to initiate a second round of cyclisation reactions which usually leads to compounds of higher structural complexity. It was already noticed in the 1950s by Ruzicka [9] and Barton and de Mayo [10], followed by a more detailed elaboration by Hendrickson [11], that 10-membered sesquiterpenes such as hedycaryol (3) can serve as neutral intermediates that can react upon reprotonation to 6-6-(selinane) or 5-7-bicyclic (guaiane) sesquiterpenes. We have recently summarised the accumulated knowledge about sesquiterpenes derived from germacrene A (2) [12] and hedycaryol (3) [13]. Now we wish to provide a review on the known chemical space of sesquiterpenes derived from germacrene B (1) (Scheme 2). Compounds Scheme 3: The chemistry of germacrene B (1). A) Synthesis from germacrone (4), B) the four conformers of 1 established by molecular mechanics calculations (energies in black boxes are relative to 1a for which the energy was set to 0.00 kcal/mol), C) Cope rearrangement to 5 and formation from 6 by pyrolysis, D) dehydration of 7 to 5 and 8. derived from 1 by oxidation will not be included in this article. The interested reader can find exemplary relevant information about this topic in references [14][15][16][17][18].
Based on molecular mechanics calculations, four conformers 1a-d have been described for 1 (Scheme 3B) [37]. The calculations revealed all four conformers are of similar stability, with 1a being the most stable conformer. The fact that 1 shows a defined set of fifteen sharp signals in the 13 C NMR spectrum [26] indicates that the interconversion between these conformers is a fast process at room temperature. This is in contrast to the findings for germacrene A (2) and hedycaryol (3) that show strong line broadening in the NMR spectra and multiple sets of peaks for different conformers [26,[38][39][40][41], pointing to a higher energy barrier between their conformers in comparison to the barriers between the conformers of 1. Like observed for germacrene A [40] and hedycaryol [41,42], 1 readily undergoes a Cope rearrangement to γ-elemene (5) above 120 °C (Scheme 3C), while the reaction of 1 with bis(benzonitrile) palladium chloride generates the palladium chloride complex of 5 from which 5 can be liberated by treatment with dimethyl sulfoxide [43]. Compound 5, with tentatively assigned structure, was first obtained as a pyrolysis product of elemol phenylurethane (6) [44]. Its structure was subsequently secured by preparation from 1 through Cope rearrangement [20] and through dehydration of elemol (7) with POCl 3 in pyridine yielding 5 and β-elemene (8) (Scheme 3D) [45]. Compound 5 has also frequently been reported from natural sources especially after heat treatment of the sample, and has been isolated from Cryptotaenia japonica [46], Bunium cylindricum [47], an unidentified Pilocarpus sp. [48], and Aristolochia triangularis [49].
Upon reprotonation germacrene B (1) can in theory yield several cyclisation products with distinct skeletons. Eudesmanes can be obtained through reprotonation at C-1 and cyclisation to intermediate I, or through reprotonation at C-4 leading to cation J (Scheme 5A). Further cyclisation modes include a reprotonation at C-4 and cyclisation to K or reprotonation at C-10 and cyclisation to L, which represent possible precursors of guaianes (Scheme 5B). For all four intermediates I-L different stereochemistries may be realised. In principle, these reactions may be enzyme catalysed or proceed without enzyme catalysis, e.g., during chromatographic purifications of compounds from complex extracts. In the latter case, because of the achiral nature of 1, racemic mixtures are expected, while enzyme products should usually be enantiomerically pure or enriched.

Eudesmanes
The eudesmane skeleton can arise by reprotonation at C-1 of 1, leading to four different stereoisomers of cation I, i.e., I1 with a trans-decalin skeleton, its enantiomer I2, I3 representing the cis-decalin skeleton, and its enantiomer I4 (Scheme 6A). In principle, the eudesmane skeleton can also be formed through cyclisations induced by reprotonation at C-4. Assuming anti addition to the C-4/C-5 double bond, these reactions lead to four stereoisomers of the secondary cation J, two with a transdecalin skeleton (J1 and J2) and two with a cis-decalin skeleton (J3 and J4). However, no natural products are known that may arise through any of these cations J, showing that a cyclisation of 1 induced by reprotonation at C-4 is not preferred. Also no compounds have been isolated with their structures rigorously elucidated that arise through cation I4. For compounds potentially generated through intermediates I1-I3 the accummulated knowledge will be discussed in the following sections.
The structure elucidation of juniper camphor (11), a compound originally isolated by chemists at Schimmel, the world leading company of the late 19th and early 20th century dealing with essential oils and perfumes, was initiated by Šorm and co-workers [75]. From the sequence of catalytic hydrogenation to 27, dehydration to a mixture of alkenes (28) and hydrogenation to selinane (29) it was concluded that 11 was a selinane sesquiterpene alcohol (Scheme 9B) [75]. Four years later, based on NMR data Bhattacharyya and co-workers suggested a cisring junction for 11 [76], but a synthesis from β-eudesmol (30) through epoxidation to 31, dehydration to 32 and epoxide opening with LiAlH 4 yielded (−)-11 (Scheme 9C) [77], contradicting this assignment.
Notably, Šorm and co-workers noticed that 11 was racemic, because neither 11 nor any of its degradation products showed optical activity [75], suggesting that the compound they had isolated arose through acid-catalysed cyclisation of 1 rather than in an enzymatic process. Also the material isolated from Platysace linearifolia showed no optical rotation [78], while the optical activity of 11 isolated from Bunium cylindricum [47], Acritopappus prunifolius [79], Aniba riparia [80], Juniperus oxycedrus [81], and Laggera alata [82] has not been determined. The ( sis during compound isolations. The reporting of (-)-11, (+)-11 and 11 of unspecified absolute configuration all under the same CAS number (473-04-1) adds to the confusion. Moreover, one report is available that mentions the isolation of 11 from Atractylodes macrocephala [87]. For unclear reason, this paper is assigned to CAS number 1647153-38-5 representing the structure of 19 (Scheme 7), which actually seems to be an unknown compound.
Compound 11 is a side product of ZmTPS7 from Zea mays [88] and 1 H and 13 C NMR data for 11 have been published [82,83]. A recent molecular docking study suggested that 11 can bind to the main protease M pro of the SARS-CoV-2 virus that is involved in viral reproduction, but experimental tests supporting this finding are lacking [89].

Eudesmanes from I2
Much less is known about sesquiterpenes derived from cation I2 (Scheme 11). The compounds described in the literature include (+)-juniper camphor (37) that can be formed by attack of water to I2. As mentioned above, this compound occurs in Cinnamomum camphora [86] and has later also been isolated from Laggera pterodonta ([α] D 24 = +4, c 0.5, MeOH) [93].

Eudesmanes from I3
Also only a few compounds potentially arising from I3 are known (Scheme 13). Compound 18 was already discussed above and can be formed by deprotonation from I1 or I3.
Cation I1 seems to be the more likely precursor than I3, because I1 is the intermediate towards structurally related natural products such as the widespread compounds 9 and 10 and a common biosynthesis of 18 through the same intermedi- ate can be assumed (Scheme 7). A 1,2-hydride shift to I3a and deprotonation could give rise to 50, a compound for which the situation in the literature is very confusing. There is no paper available describing the isolation and structure elucidation of a compound with the structure of 50, and the first published paper that can be found under the CAS number of 50 (869998-21-0) does not mention this compound [100]. Several later reports claim the detection of "eudesma-5,7(11)-diene", a name assigned to CAS number 869998-21-0, but neither a structure is shown nor a reference to previous work is given in these reports, leaving doubt about the stereostructure the authors of this work had in mind [101][102][103]. One recent report mentions the detection of "eudesma-5,7(11)-diene", but again no structure is shown, and the structural assignment is based on a com- parison of retention indices [104]. However, the deviation between measured and reference retention index is quite large (I = 1572 vs 1543), and the reference data originate from [103] in which the basis for structural assignment is unclear. Finally, one more paper assigned to CAS number 869998-21-0 mentions the detection of "eudesma-5,7(11)-diene", but in this case the structure of 38 (Scheme 11) instead of 50 is shown, which based on a comparison of the measured to a database retention index may at least in terms of the relative configuration be a correct structural assignment [105]. Taken together, the confusing situation for 50 in the literature demonstrates impressively, how inaccurate data reporting can lead to unclear structural assignments and even error propagation, and shows the importance of structure elucidation by classical methods, i.e., isolation and compound characterisation by NMR spectroscopy and determination of optical rotation.
Compound 51 can be generated biosynthetically from I3a through 1,2-methyl migration to I3b and deprotonation. However, this hydrocarbon has not been isolated from natural sources and is only known as racemic synthetic material [106].
Similarly, 52 has only been described as a synthetic compound [107]. Its hypothetical biosynthesis is possible from I3a by Wagner-Meerwein rearrangement to I3c and deprotonation.

Guaianes
As discussed above, the cyclisation of 1 induced by reprotonation at C-4 to the eudesmane skeleton encounters obstacles because of the formation of secondary cations. Preferentially, reprotonation at C-4 leads to the guaiane skeleton since the formed cations are tertiary. Alternatively, reprotonation of 1 at C-10 can also induce the formation of the guaiane skeleton.
The guaiane sesquiterpenes derived from cationic intermediates K1, K2 and K4 are summarised in Scheme 15A, while no compounds are known whose formation could be explained from K3. β-Bulnesene (53), a product by the deprotonation of K1 or K2, was first isolated from the guaiac wood oil of Bulnesia sarmientoi [108] and later also observed in Pogostemon cablin [109]. Bulnesol (57), a compound of known absolute configuration [110] that occurs in the same essential oil [108], has been converted through pyrolysis of its acetate 58 into 53 (Scheme 15B) [111], securing the relative configuration. This work did not comment on the question of absolute configura- tion, but assuming a common biosynthesis of 53 and 57 analogous absolute configurations for these compounds are likely.
The guaiane sesquiterpenes that are potentially derived from cationic intermediates L1-L4 are summarised in Scheme 16A. trans-β-Guaiene (54) can either be generated from K1 undergoing a 1,2-hydride shift to K1a followed by deprotonation (Scheme 15A), or from L4 through a similar sequence of steps (Scheme 16A). Its enantiomer ent-54 could analogously arise from K4 or L1. The first detection of this compound was claimed from Aframomum alboviolaceum, but this study did not report on the isolation and structure elucidation [117]. Rather the identification was only based on GC-MS data, without a reference to a previous identification through rigorous structure elucidation. Conclusively, this compound has not been described thoroughly and its identification is doubtful. Information about the mass spectrum and Kovats retention index have been added to data bases such as the NIST Chemistry Webbook [118], which promoted the ambiguous detection of 54 in many other species, as described in more than 300 papers to date.
Compound 55 can be formed from K2 through capture with water. A compound with the same planar structure of 55* named guai-7(11)-en-10-ol has been reported from Zanthoxylum syncarpum with fully assigned 1 H and 13 C NMR data, but unresolved relative and absolute configuration [119]. by Šorm and co-workers [123]. It is well known that 56 can easily be dehydrogenated, e.g., by heating with sulphur, to the blue azulene derivative 62 (Scheme 16C) [121,122,[124][125][126], but the structure elucidation of this compound was only completed in 1936 [127]. Based on a comparison of IR spectra of natural terpenes, their hydrogenation and dehydrogenation products, the correct planar structure of 56 was concluded by Pliva and Šorm [128]. After the absolute configuration of 61 was solved [129], the full stereostructure of 56 became known.
No total synthesis and no NMR data are available for 56. β-Guaiene is one of the main constituents of the essential oil from Achillea millefolium that shows inhibitory activity against Babesia canis, a parasite transmitted by ticks that infects blood cells [130].

Conclusion
As summarised in this review, the biosynthesis of many sesquiterpene hydrocarbons and alcohols exhibiting the eudesmane or guaiane skeleton can be explained from the neutral intermediate germacrene B, although not all compounds known to literature have been isolated from natural sources; some com-pounds are only known as synthetic materials. Compared to the known compounds arising from germacrene A or hedycaryol through similar reactions as discussed here [12,13], however, the number of terpenes derived from germacrene B is much lower. In this article we have explained the rationale for the structure elucidation including relative and, if known, absolute configurations. Through a detailed analysis of the available information it also turned out that some of the assigned structures are doubtful. The importance of rigorous structure elucidation, historically usually performed by chemical correlations and today preferentially done by NMR spectroscopy or X-ray analysis, is clearly evident from the fact that wrongly reported structures or structures assigned without any comprehensible basis lead to error propagations and highly confusing situations in the literature. Today many reports are only based on tentative GC-MS assignments, often even without comparison to authentic standards, which results in a lot of information of questionable relevance. The large number of such papers published today makes it more and more difficult to find the relevant information in the literature. With this work we hope to help the interested reader to have an easier access to the knowledge about sesquiterpenes derived from germacrene B.